Journal of Pharmaceutical Negative Results

: 2011  |  Volume : 2  |  Issue : 2  |  Page : 115--120

Ultra violet spectrophotometric method: Not possible for the simultaneous estimation of alpha one adrenoreceptor blockers

Alankar Shrivastava1, Vipin Bihari Gupta2,  
1 Jodhpur National University, Jodhpur, Rajasthan, India
2 BR Nahata College of Pharmacy, TIFAC-CORE Innovation square, Mhow-Neemuch Road, Mandsaur, India

Correspondence Address:
Alankar Shrivastava
Department of Pharmaceutical Analysis, TIFAC-CORE Innovation Square, B.R. Nahata College of Pharmacy, Mhow-Neemuch, Mandsaur (M.P)


Background: Although initially introduced for the management of hypertension, al-adrenergic-receptor antagonists (a 1 blockers) have become the standard of care for the medical management of benign prostatic hyperplasia (BPH)-related lower urinary tract symptoms (LUTS). Alpha-blockers (alfuzosin, tamsulosin, doxazosin, prazosin, and terazosin) relax the smooth muscles in the prostate and are indicated for the symptomatic treatment of BPH, due to evidence of their positive and rapid effect on LUTS. Objective: Our objective was to develop and validate a method for simultaneous estimation of these drugs. Our aim was to develop a UV spectrophotometric method because of the simplicity and economical advantage of this technique. Materials and Methods: All these drugs were dissolved in the same solvent (methanol) and scanned under a ShimadzuUV spectrophotomer, under full UV (ultraviolet) range (200 - 400 nm). Result: After some experiments we found that this is impossible. This was evident from our study that UV spectra were overlapping each other, except in the case of tamsulosin. Conclusion: Same class of drugs may have almost the same functional groups, and gradient Reverse-Phase Liquid Chromatography(RPLC) will be more useful to separate such complicated mixtures. Therefore, we propose to develop a gradient High-Performance Liquid Chromatography(HPLC) method as it may be a more suitable method for the simultaneous estimation of these drugs.

How to cite this article:
Shrivastava A, Gupta VB. Ultra violet spectrophotometric method: Not possible for the simultaneous estimation of alpha one adrenoreceptor blockers.J Pharm Negative Results 2011;2:115-120

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Shrivastava A, Gupta VB. Ultra violet spectrophotometric method: Not possible for the simultaneous estimation of alpha one adrenoreceptor blockers. J Pharm Negative Results [serial online] 2011 [cited 2020 Jan 29 ];2:115-120
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Benign prostatic hyperplasia (BPH), a condition characterized by hyperplastic nodules in the periurethral region and transition zone of the prostate, overall prostatic enlargement, and lower urinary tract symptoms, is highly prevalent among middle-aged and elderly men. BPH is rarely life-threatening, but it can lead to acute urological problems, for example, acute urinary retention (AUR). The clinical manifestations of BPH include LUTS, poor bladder emptying, urinary retention, detrusor instability, urinary tract infection, hematuria, and renal insufficiency. The prevalence of histologically identifiable BPH for 60-year-old males is greater than 50% and approximately 90% by age 85. One out of every four men in the United States is being treated to relieve the symptoms of BPH by age 80.

The causes of BPH are unknown; aging and the presence of male androgens are the dominant risk factors. Obtaining a medical history is a key step in assessing whether lower urinary tract symptoms are due to BPH or some other process. Optimal decisions about treatment are best made by a clinician and an informed patient working together. BPH is treated primarily to improve the quality, rather than increase the quantity of life. [1]

α1 -Adrenoceptor blockers role in benign prostatic hyperplasia and in prostrate apoptosis [1]

α1 -Adrenoceptors belong to the superfamily of G-protein-coupled adrenergic receptors, which mediate the actions of endogenous catecholamines (norepinephrine and epinephrine). Two types of α1 -adrenoreceptors have been identified in the bladder and at the bladder neck and in the prostatic smooth muscle surrounding the urethra. Targeting these receptors as a means of preventing smooth muscle contraction givesrise to a class of medications commonly used to treat LUTS. There are currently four recognized subtypes of α1-adrenoceptors identified in the prostate: α1a, α1b, α1d, and α1L. Classically, the α1a -adrenoceptor antagonists have been used to target the stromal smooth muscle cells in the treatment of BPH, in order to reduce their tone. The competitive inhibition of catecholamines prevents smooth muscle contraction by interfering with the perpetuation of second messenger pathways and subsequently modulating cytoskeletal proteins in the prostatic smooth muscle cells. The therapeutic efficacy of α1-adrenoreceptor antagonists for the treatment of BPH has been clinically documented and the safety profiles of these α1 -blockers are now well-established as a result of their long-term use as front-line antihypertensive agents.

A model outlining the potential action of quinazoline-based α1 -adrenoceptor antagonists [Figure 1] on apoptosis signaling pathways in prostate cancer cells,has also been proposed. Transforming growth factor-β (TGF-β) signaling involves formation of the receptor complex, Smad activation, nuclear translocation, and gene transcription that subsequently leads to apoptosis. Cell survival or mitogenic pathways initiated by activated protein tyrosine kinases (PTK) can repress this pathway at different levels and may inhibit TGF-β signaling. Asstrong evidence suggests that quinazoline-based drugs can prevent PTK activation, doxazosin (as a quinazoline-based α1 -adrenoceptor antagonist) could potentially uncouple these pathways to facilitate apoptotic signaling by TGF-β. Considering that apoptosis induction in prostate biopsies from BPH patients was observed over the normal dose range, effective application of these FDA-approved agents, the quinazoline-based α1 -antagonists, may have promising therapeutic effects, not only in the long-term management of BPH, but also forprostate cancer, potentially resulting in decreased morbidity. However, this communication does not give any information about prazosin, which also contains a quinazoline nucleus in its molecular structure.{Figure 1}


Prazosin1-4 (amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furanylcarbonyl) piperazineHCl [2] is official in the British Pharmacopeia, [3] Indian Pharmacopoeia, [4] United States Pharmacopoeia, [5] and European Pharmacopoeia. [6]Prazosin, a short-acting α1-specific antagonist requiring multiple daily doses, was investigated in the treatment of BPH as a result of its specificity for the α1 -receptor. [1]


Terazosin hydrochloride dehydrate RS-1-(4-amino-6,7- dimethoxy-2-quinazolinyl)-4-[(tetra-hydro-2-furanyl) carbonyl]-piperazine monohydrochloride, [6] a once-a-day specific α1 blocker was approved by the food and drug administration in 1987, for the treatment of hypertension, and in 1993, for the treatment of BPH. Terazosin has been shown in multicenter, placebo-control trials to improve the symptom scores. [7] This drug is official in European Pharmacopoeia. [6]


Doxazosine[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]-(2,3-dihydro-1,4-benzodioxin-2-yl)methanone [6] is a quinazoline compound of the methane sulfonate family; peak plasma concentrations are achieved within two to three hours of` ingestion, with a bioavailability of 65%. [7] Doxazosin is the second α1 -blocker approved by the FDA for the treatment of symptomatic BPH. The potential advantage of doxazosinis its longer half-life tolerability, but on the basis of its comparable efficacy and tolerability to terazosin,its longer half-life does not seem to confirm any clinical advantage. [8] Doxazosin is official in European Pharmacopeia. [6]


Tamsulosin hydrochloride (TAM), (-) - (R) - 5 -[ 2- [[ 2-(o-Ethoxyphenoxy) ethyl] amino ] propyl ] - 2-methoxy benzene sulfonamide monohydrochloride [10] is a subtype-selective α1a and α1d-adrenoceptor antagonist. α1 receptors predominate in the prostate gland, prostatic capsule, prostatic urethra and bladder. The relaxation of prostate and bladder smooth muscles is associated with improved maximal urine flow (Q max ) and alleviation of lower urinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia (BPH). [11]Tamsulosin blocks α1a and α1d, with a ten-fold higher affinity than it, blocks the α1b adrenoreceptors, and is therefore considered to be α1a subtype selective. [12]Tamsulosin clearly offers advantages over other α1 adrenoceptor antagonists in terms of the need for a single daily dose only, and its low potential for hypotensive effects or interference with concomitant antihypertensive therapy. Dosage titration at the start of treatment is not necessary. Tamsulosin has a rapid onset of action and is effective in patients with moderate or severe symptoms. The drug is therefore a valuable therapeutic option, with both demonstrated and potential advantages over older nonselective agents, forthe management of patients with LUTS associated with BPH. [11]


Sustained release alfuzosinN-[3-[(4-amino-6, 7-dimethoxy-quinazolin-2-yl)-methyl-amino]propyl] oxolane-2-carboxamide hydrochloride [13] is the fourth α1 -selective blocker approved by the FDA for the treatment of symptomatic BPH. [9] The efficacy and safety of a new once-daily (OD), extended-release, formulation of alfuzosin (alfuzosin OD) have been demonstrated in two pivotal double-blind Phase III studies, and summarized in a pooled analysis of three studies. Studies of the immediate release formulation had previously demonstrated significant effects on the Q max as early as 1.5 hours after the initial dose. [14] Human studies also suggest that alfuzosin OD exhibits a urodynamically measurable effect on bladder outlet obstruction due to benign prostatic hyperplasia, in men with lower urinary tract symptoms within hours of the first administration [Table 1]. [15]{Table 1}

Literature review for the methods available in the literature for the determination of these class of drugs are already published in the Current Pharmaceutical Analysis Journal, [1] with highlights on various analytical methods published on alphaone blockers with comparison in terms of sensitivity, language, presentation, applications, advantages, and disadvantages. Twelve spectrophotometric and 39 chromatographic methods including,two ESI MS / MS, one ESI MS, two HPTLC, three TLC, seventeen HPLC with UV, nine with fluorescence, one with electrochemical detection, and four with MS detector, were found. Other than this, threevoltametry, onemethod each for radioreceptor assay, polarography, capillary electrophoresis, and potentiometry titrations were found. Radioreceptor assay, LC-MS, and the UPLC-MS methods were found to be the most sensitive for prazosin, terazosin, and doxazosin, respectively. Likewise, HPLC-MS-MS methods were found to be themost sensitive methods for the estimation of tamsulosin and alfuzosin.

Surprisingly, method for the simultaneous separation of all alpha one blockers is not reported in literature. The proposed method reduces the time required for switch over of chromatographic conditions, equilibration of the column, and post column flushing that are typically associated when different formulations are analyzed. Any of the drugs can be used as an internal standard for the estimation of another drug.

Therefore, simultaneous estimation of these alpha one adrenoreceptor blockers was attempted and development of the UV spectrophotometric method was aimed for, initially because of its simplicity and economical characteristic, as compared to the HPLC method. However, soon we found that it is not possible to develop a simultaneous spectrophotometric method for these drugs. The reasons and the outline of the possible method are described in this communication.

 Materials and Methods

Chemicals and reagents

Doxazosin and Alfuzosin were received fromDr. Reddy Laboratories,Tamsulosin was received from AurobindoLaboratories. Prazosin and Terazosin were obtained from ArtiLaboratories and Sun Pharma, respectively. Methanol used was of AR grade, purchased from Lobachemie Laboratories.

The UV spectrophotometer of Shimadzu Corporation Limited, model no. 1800, with 1 cm path length of sampling cells, was used for the present study.

For the applicability of the simultaneous equation method [26] some criteria must be fulfilled. The criterion is:

The ratio:


should lie outside the range of 0.1 - 2.0. These criteria are satisfied only when two components are reasonably dissimilar.

The information required was:

The absorptivity of the first drug at λ1 and λ2 , that is, a x1 and a x2 , respectively.The absorptivity of the second drug at λ1 and λ2 , that is, a x1 and a x2 , respectivelyThe absorbance of the diluted sample at λ1 and λ2 ,that is, A 1 and A 2 , respectively.

Preparation of sample

Fifty milligrams of each drug, accurately weighed,was transferred into a 50 ml volumetric flask and diluted upto the mark with methanol. Prepared solution of 0.5 ml was again transferred to a 50 ml volumetric flask and diluted up to the mark with methanol. These solutions (10 ΅g / ml) were scanned under the UV region from 200 to 400 nm. [Figure 2] represents an overlay of the spectra of five alpha one adrenoreceptor blockers under study.{Figure 2}

It is clear from [Figure 2] that it is practically impossible to locate λ1 and λ2 in the case of simultaneous estimation of pairs of four drugs; prazosin, terazosin, alfuzosin, and doxazosin. Further Tamsulosin shows some absorbance at the λmax of the other four drugs.


In this manner we attempted to develop the UV spectrophotometric method for the simultaneous estimation of prazosin, terazosin, doxazosin, tamsulosin, and alfuzosin.

It was observed that simultaneous estimation was not possible because of the overlapping of the spectra of all drugs, except tamsulos in [Figure 2]. HPLC was the dominant separation technique in modern pharmaceutical and biomedical analysis, because it resultedin highly efficient separations and in most cases provided high detection sensitivity. Chromatographic optimization procedures are becoming more multidisciplinary, as methods are sought to obtain a lot more information on the separations, which may be isocratic or gradient.

It is apparent from the present considerations that the goal of fast chromatography can best be approached in liquid chromatography by employing gradient elution. Gradient elution RPLC is a powerful technique required to separate samples that otherwise exhibit the general elution problem under isocratic conditions. Delivering competent analytical judgment on samples in a timely manner is becoming more difficult as the sample load in quality control laboratories continues to increase.

Gradient elution gave a shorter overall analysis with similar resolution of the critical pair compared to isocratic elution, without sacrificing repeatability in retention time, peak area, and peak height or linearity of the calibration curve. The main factors that drive chromatographers to use gradient elution are multi-component samples, which span a wide range in retention. [27] Hence, we propose to develop the gradient HPLC method for the simultaneous estimation of these drugs.


The presented study is part of the research study donefor the award of Doctor of Philosophy from the Jodhpur National University. The authors acknowledge TIFAC-CORE, a scheme of the Department of Science and Technology (DST), Government of India, for providing the UV instrument [Ref. no. TIFAC/MR/CORE/BRN/2008, dated 07/11/2008] to our institute, through which this study became possible. We are also thankful to the B.R. NahataCollege of Pharmacy for providing support in terms of solvents and supporting staff.


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